The objective of this research is to learn more about how osteopontin (OPN) promotes the survival of mammalian cells exposed to xenobiotic or cytokine injury, particularly with regard to N0 production. The principal investigator's goal is to find ways to use this information to control N0 production, inflammation and toxicity in clinically important situations, for example after exposure to high levels of environmental or man-made toxins. N0 is an important second message; it is implicated among other things in controlling vascular tone, mediating cell killing by certain cytotoxic cells, and conveying signals. Because of the potentially lethal impact of N0 on cells, it is critical that its production be subject to close regulation. OPN appears to control the level of N0 and perhaps other free radicals and oxidants via a protein phosphorylation signaling pathway that regulates key transcription factors and modifies intracellular calcium levels. Specific aims are: 1) To test the hypothesis that inhibition of activation of the transcription factor Nfk B by OPN accounts for the inhibition of lipopolysaccharide and interferon-g-induced iN0S (inducible nitric oxide synthase, N0sII) transcription in macrophages; 2) To determine whether OPN-induced protein phosphorylation or decreased intracellular free [Ca plus plus] underlies the inhibition by OPN of angiotensin II-stimulated N0 production by the constitutive N0S in kidney proximal tubule cells; 3) To clone the mRNA encoding iN0S from a neuroepithelial cell line that appears to make an unusual N0S protein in order to determine whether the mRNA is spliced in a novel fashion; 4) To evaluate the hypothesis that in certain situations OPN can protect cells from the cytotoxic (or cytostatic) action of a particular agent (keratinocytes exposed to TPA or inflammatory mediators; neurons exposed to astrocytes activated by g IFN plus LPS to produce N0). We will determine whether OPN can inhibit the induction of N0SII transcription in hepatic endothelial cells exposed to dioxin (TCDD), and we will assess the ability of OPN to reduce tissue damage in mouse skin exposed to ultraviolet light B radiation or to the phorbol ester TPA, a protein kinase C activator. To investigate on mechanism OPN may use to promote cell survival, its ability to alter the expression of genes known to be involved in programmed cell death will be studied. 5) To examine in the intact animal the ability of OPN to ameliorate acetaminophen-induced liver injury and relate this to a suppression of iN0S expression. Parts of the last two specific aims will exploit their recently developed knockout mouse that is unable to make OPN. Some of the methods employed include DNA band-shifts with Nfk B probes, analysis of Ca plus plus levels with Fluo-3 using a confocal laser-based scanning microscopy, northern and western analyses of gene expression, histology and immunohistochemistry.